Detection Of Listeria Monocytogenes In Cheese And Samples Of The Production Environment: Comparison Of The One-Step Enrichment Broth “OneBroth™“ With Reference Method ISO

(1)

CH-3003 Berne www.alp.admin.ch

joerg.hummerjohann@alp.admin.ch

Detection Of Listeria Monocytogenes In Cheese And Samples Of The Production Environment: Comparison Of The One-Step Enrichment Broth “OneBroth™“ With Reference Method ISO 11290-1

J. Hummerjohann, D. Weik, B. Ulmann, R. Imhof

For Listeria monocytogenes (LMO), two-step enrichment procedures gave the most satisfiying recovery rates in all relevant food matrices so far, but they are still time-consuming. Recently, a procedure called “One Broth (TM)” containing a single enrichment step with 24 hour incubation was successfully validated against the reference method ISO 11290-1/Amd:2004.

However, before implementing an alternative method, one has to verify the performance, especially on matrices which are often used or known to be problematic for LMO recovery . Results of our verification procedure are reported here.

ENRICHMENT:

1) Mix 10 g sample & 90 ml „One Broth™“ (Oxoid) 2) Incubation at 30°C for 22 -26 h

PLATING OUT AND CONFIRMATION:

1) ALOA (Biolife), 37°C, 24-48 h; 2) confirm 1-5 blue colonies with halo per plate with gene probe (Accuprobe, Biomerieux) 2nd ENRICHMENT:

Fraser broth, 37°C, 46-50 h

Methods*: „One Broth™“ ISO 11290-1/Amd:2004

The one-step enrichment procedure „One broth™“ is suitable for the fast and successful recovery of Listeria monocytogenes in cheese and samples from the production environment, but may need modification in case of certain matrices (e.g. 48 h incubation of brine samples). These results are showing the need of a carefully performed verification procedure, even when already validated alternative methods are applied.

* deviations from AFNOR validation study or ISO 11290 method: 10g sample; ALOA agar for all experiments ; no second agar; Accuprobe is a validated alternative confirmation test

1st ENRICHMENT:

1) Mix 10 g sample & 90 ml ½ Fraser Broth 2) Incubation at 30°C for 22 -26 h

ISO 11290

+ -

^Σ

+

⁵⁰⁰ ¹¹ ⁵¹¹

One Broth ™

-

³¹ ³² ⁶³

Σ 531 43 574 = n

cfu/sample OneBroth +ve of 15

ISO 11290-1 +ve of 15

7 7 14 (1)*

35 14 14 (8)*

LMO 1/2a

485 15 15 (14)*

3 6 15 (3)*

28 15 15 (10)*

LMO 4b 402 15 14 (14)*

3 LMO + 7 LIN 8 15 (5)*

28 LMO + 82 LIN 15 15 (8)*

Brine Samples Spiked with L. monocytogenes & L. innocua LMO 4 b & LIN

402 LMO +680 LIN 15 15 (15)*

Design of spiking experiments:

• smear water, brine (20% NaCl), red smear soft cheese, hard cheese smear analysed after scheme above

• 3 levels of contamination (low, medium, high)

• 3 strains, isol. from cheese (LMO 1/2a, LMO 4b & L. innocua)

• 15 samples per contamination level and strain (e.g. in Tab. 1)

• 5 blanks/food type

And 14 naturally smear water contaminated samples

Tab. 1: Design of spiking experiments and results (brine samples)

* in (): samples positive from plating out after first enrichment

Results:

• For brine samples spiked with low levels, only 21/45 positive with

„One Broth™“ in contrast to reference (44/45 positive) (Tab.1)

• Second enrichment of ISO 11290 strongly needed for LMO recovery from brine samples (Tab. 1)

• Clear concordance between both methods (kappa=0.57, Tab 2);

strong concordance (kappa=0.71), if brine sampels excluded

• Successful recovery of LMO at low numbers in brine, if „OneBroth™“ is incubated for 48 h (data not shown)

Tab. 2: Summary of method comparision

Clear concordance between both methods (kappa=0.57) , calculated after Swiss SAS guideline No. 328